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Myelinic nanochannels in neurodegenerative diseases

Periodic Reporting for period 3 - MyeliNANO (Myelinic nanochannels in neurodegenerative diseases)

Reporting period: 2019-04-01 to 2020-09-30

"Overall goal of the ERC Advanced Grant MyeliNANO is a better understanding of oligodendrocytes and myelin in the aging central nervous system. We are combining advanced mouse genetics, biochemistry and cell biology, novel metabolic imaging, advanced electron microscopy and behavioural/clinical analyses to study the role of forebrain myelination in neuropsychiatric diseases and the function of nanometer-wide cytosolic channels within myelin (hence ""MyeliNANO"") for the metabolic support of functional axonal integrity and long-term survival of myelinated axons. This line of research will identify age-dependent risk factors for major neurodegenerative reasons that have been recognized as major socio-economic burden of an increasingly aging society."
"Main achievements in the first half of the funding period according to the original description of action plan in part B2 of the application are as follows:

Part I: Studying higher brain functions and plasticity in myelin mutant mice

1. Forebrain-specific targeting of myelin protein genes
- We have successfully generated Mbpflox/flox mice for the targeted prevention of myelination by cell-specific and/or inducible Cre recombination (Meschkat et al., manuscript in preparation).
- As an extension to the proposal we have also added floxed Mrf mice to our experiments, because upon Cre recombination they are arrested at the OPC stage.
- By cross-breeding Mbpflox/flox mice to the Emx1-Cre driver line, we obtained mice lacking cortical myelination and corpus callosum myelination in the forebrain (Meschkat et al., manuscript in preparation).
- As an extension to the proposal, we have also generated FoxG1-IRES-Cre:: Mbpflox/flox mice lacking myelination in the entire forebrain. The latter is likely the better model for behavioural studies. Neither conditional Mbp mutant mouse exhibits the disruptive motor phenotype of shiverer mice that prevented behavioral analyses in the past.

2. Inducible arrest of adult myelination: targeting adult oligodendrocyte precursors (OPC)
- We have successfully generated Plp1-CreERT2::Sip1flox/flox mice for the inducible prevention of new myelin synthesis in the adult brain. While aiming for the analysis of adult myelination in cognitive functions, we have also tested the role of adult myelination motor learning on the complex running wheel, a prior ""bench mark"". Surprisingly, our observations of unimpaired motor learning are at odds with the published findings of inducible Myrf1 KO mice, which reportedly exhibit impaired motor learning. This may reflecting differences of the myelination-associated target genes of the two transcription factors Sip1 and Myrf1 in oligodendrocyte precursor cells. (Goebbels et al., unpublished).

Part II: Myelinic nanochannels: a determining factor of brain aging and neurodegenerative diseases?

3. Injury to myelinic nanochannels in MS-relevant models of neuroinflammation
- We have shown that Cnp KO mice loose myelinic nanochannels in myelin, as CNP and MBP anatagonize each other. In the absence of CNP, MBP can cause myelinic channels to collapse (Snaidero et al., 2017).
- We found that CNP-deficient mice exhibit unique behavioral abnormalities, best described as catatonia-like postural defects. They are caused by secondary inflammation as demonstrated by the pharmacological depletion of microglia, which rescues this phenotype (Janova et al., 2018; Garcia-Agudo et al., 2019).
- We have successfully performed EAE experiments in Plp-transgenic and Mbpneo/neo hypomorphic mice, both with mosaic dysmyelination of the spinal cord. We found that the presence of naked axons ameliorates the EAE phenotype without changing the immunization profile. This supports our hypothesis that myelin does not shield axons from the effects of neuroinflammation, but rather is its primary target. Myelin under (auto)-immune attack becomes a major risk factor of axonal degeneration in inflammatory disease models (Stassart et al., in preparation).

4. Protein deposits within myelinic nanochannels in mouse models of neurodegenerative disease
- We found that the Cnp-Cre mediated deletion of the SOD transgene from floxed ALS mice ameliorates the clinical phenotype, pointing to a role of oligodendrocytes in this disease model (A. Mot; unpublished data)
- We have studied SOD transgenic mice with the working hypothesis that abnormal SOD aggregates, when forming within myelinic nanochannels, will aggravate the (ALS-like) disease in the rodent model. Indeed, in the absence of CNP the ALS course of disease is accelerated. We think this is because reduced myelinic channels (Snaidero et al., 2017) make it more likely that SOD aggregates hinder the metabolic support of myelinated axons (Mot et al., in preparation).
- We have purified myelin from SOD transgenic mice and can readily demonstrate SOD by Western blot analysis, demonstrating the association of SOD with the myelin membrane fraction, in good agreement with our working hypothesis (Mot et al., in preparation).
- By electron microscopy, we have been able to visualize SOD aggregates within the cytosolic compartment of myelinating oligodendrocytes (Mot, Moebius et al., in preparation).

5. Impact of reduced myelinic nanochannels on proteolytic processing of APP
- We have successfully generated Alzheimer using mouse models with genetically-induced advanced white matter aging. In our hypothesis, myelin aging and structural abnormalities are the major age-associated risk factor for AD because they collectively trigger the BACE1-dependent Abeta cascade. Soon to be published data demonstrate that these 5XFAD::Cnp-/- mice exhibit indeed an earlier deposition of cortical and white matter plaques. A detailed characterization of this new AD model is in progress (Depp et al., manuscript in preparation).
MyeliNANO comprises experimental and conceptual aspects that are completely 'beyond state of the art', as indicated in the summaries above. For example in part I of the project, this is the first demonstration that cortical myelin is required for information processing, in addition to its known function in speeding conduction This novel function involves the newly discovered axonal metabolic support by oligodendrocytes (Moore et al., Nat Comm, 2020). It will soon be complemented by the behavioural analysis of mice that selectively lack intracortical myelin in the forebrain.

In part II of the project, the hypothesis-driven discovery that myelin itself that can be mobilized upon glucose withdrawal has the potential to completely change our view on white matter as a previously overlooked giant energy reserve in the brain. This study will very soon be submitted to a major journal.